PRIMARY STRUCTURE OF THE 5-S SUBUNIT OF TRANSCARBOXYLASE AS DEDUCED FROM THE GENOMIC DNA-SEQUENCE

Transcarboxylase from Propionibacterium shermanii is a complex biotin- containing enzyme composed of 30 polypeptides of three different types . It is composed of six dimeric outer subunits associated with a centr al cylindrical hexameric subunit through 12 biotinyl subunits; three o uter subunits on each face of the central hexamer. Each outer dimer is termed a 5 S subunit which associates with two biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl-CoA and p yruvate form propionyl-CoA and oxalacetate, the 5 S subunit specifical ly catalyzing one of these reactions. We report here the cloning, sequ encing and expression of the monomer of the 5 S subunit. The gene was identified by matching amino acid sequences derived from isolated auth entic 5 S peptides with the deduced sequence of an open reading frame present on a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3 S biotinyl subunit. The cloned 5 S gene encodes a protein of 519 amino acids, M(r) 57,793. The deduced sequence shows regions of extensive homology with that of pyruvate carboxylase and o xalacetate decarboxylase, two enzymes which catalyze the same or rever se reaction. A fragment was subcloned into pUC19 in an orientation suc h that the 5 S open reading frame could be expressed from the lac prom oter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots which co-migrated with authen tic 5 S and were fully active in catalyzing the 5 S partial reaction. We conclude that we have cloned, sequenced and expressed the monomer o f the 5 S subunit and that the expressed product is catalytically acti ve.